Thymidylate synthase (TS) is a key target for chemotherapy of colorectal cancer and other solid tumors. 5-fluorouracil, the drug of choice for treatment of metastatic colorectal cancer has inhibition of TS as a major site of action after metabolism to the corresponding deoxynucleotide. Because of its role as a target of drug action and as a key enzyme involved in DNA biosynthesis, the TS gene and its regulation have been topics of substantial investigation. Regulation of TS gene expression in human and murine cells has been demonstrated to be post-transcriptional by others. It has now been determined that human cells contain RNA species which are complementary to portions of the human thymidylate synthase gene and mRNA. There are at least two distinct types of antisense RNAs. A cDNA clone corresponding to a TS antisense RNA (3'rTS RNA) has been obtained and characterized. 3'rTS RNA is polyadenylated, apparently spliced and overlaps with the 3'noncoding region, exon 7 and a portion of intron 6 of the human TS gene. This is the first demonstration of an antisense RNA which overlaps a human gene and is both polyadenylated and apparently spliced. It is proposed that the TS antisense RNAs be more fully characterized and their identity as playing a role in TS gene expression be determined. The possible interaction of these sense and antisense gene transcripts will be studied. It is also proposed that if a role of TS antisense RNAs in TS gene expression is found, the relevance to exposure of cells to TS inhibitors be investigated to determine if this can be exploited to improve cytotoxicity.